Friday, 30 September 2011

Techniques in Molecular Biology: Monoclonal Antibody Production

Antibodies are perhaps one of the most important tools in the arsenal of molecular biologists. They have a wide variety of applications, from targeting macromolecules with fluorescent dyes or other indicators to aid in visualization, or as a component of other molecular techniques, such as immunopercipitation of proteins. Antibodies that specifically target a researcher's molecule of interest, though, have not always been in the biologist's toolbox.

Before researchers had this ability, antibodies were produced using a simple technique: inject your antigen of choice into a mouse (or goat, or rabbit), and the mouse will produce antibodies targeted against the antigen. After taking a blood sample from the mouse and collecting the serum, the antibodies could be purified. Antibodies produced in this manner were said to be polyclonal: that is, they were derived from multiple antibody producing B-cells in the spleen. Each antibody producing cell produces a different antibody1, and antibodies produced in this way will be a mixture of different antibodies from different cells.

Monoclonal antibodies, in contrast to the polyclonal variety, are derived from a single antibody producing cell. Antibodies from a single cell will all be specific for the same antigen epitope. This confers several advantages over polyclonal antibodies. Perhaps the biggest advantage is that they allow researchers to target specific epitopes on an antigen. Let's say you wanted to mark a specific residue on a protein using antibodies that are labelled with a fluorescent dye. Using polyclonal antibodies, your protein would end up labelled all over, since the polyclonal antibodies would bind of a variety of epitopes on the protein's surface. Monoclonal antibodies specific to the residue of interest would get rid of all the problematic non-specific binding.

The production of monoclonal antibodies, however, is somewhat different, and pretty cool. The technique starts off just as it would if you were making polyclonal antibodies: inoculate a mouse using your antigen of interest. The antibody producing B-cells in the mouse spleen will begin to make antibodies that target your antigen. Where Köhler, and Milstein's technique differs is in what is done with those B-cells. Normally, the antibody-producing cells only start making antibodies near the end of their life. Isolating individual cells (thus isolating only one type of antibody) and growing them in culture would work to produce monoclonal antibodies, but only for a short amount of time. Milstein and co. got the idea of fusing the B-cells with immortalized mylenoma cells. Cancer cells can, for a variety of reasons, become immortalized2, and continue replicating - indeed, this is what makes cancer a problem! By creating hybrids between B-Cells and mylenoma cells - called hybridomas - they were able to created antibody-producing cells that live forever and keep on producing antibodies. Culturing these cells and purifying the antibodies now became a more viable option. Separate cell lines were isolated and cultured, so that each culture contained only cells from an individual lineage, and consequentially, produced only one kind of antibody. These cells are first grown on plates to establish a lineage, but are eventually transferred to large tissue culture flasks. This allows for tons of monoclonal antibodies to be produced, isolated, and used by researchers the world over.

This technique pioneered by Milstein and Köhler revolutionized the way research is done in molecular biology. It was important enough that it won Georges Köhler, and César Milstein, the Nobel Prize in Medicine in 19843. Since then, antibodies that target any antigen imaginable have been developed, and can easily be ordered from companies that specialize in production of monoclonal antibodies. It is unlikely, I think, that monoclonal antibodies will be ever displaced as a staple tool for molecular biology research.

FURTHER READING:

I you're interested in reading more about the history of antibodies, their discovery and the story of Milstein and Köhler's work, I suggest reading through "A Brief History of the Antibody" posted at the Proteintech Group blog [Part IPart IIPart III]. Milstein's Nobel lecture also contains some interesting insights into antibodies, antibody research and the development of Hybridoma technology, and can be found here.

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1. The different antibodies produced are specific for the same antigen, but are directed towards different epitopes on the antigen.

2. This is to say that they are not subject to the Hayflick Limit. The Hayflick Limit, named after American researcher Leonard Hayflick, is the number of times a given cell line can divide before stopping. Originally, cells were thought to replicate indefinitely, and failure to keep cell lines alive was thought to be due to ignorance of optimal techniques. Hayflick and Paul Moorhead, working at the Wistar Institute in Philadelphia in 1961, showed experimentally that cell lines impose a limit on the number of times they can divide. The limit differs between cell types, but for humans it is around 52 divisions.

3. The prize was shared with Neils Jerne, who was awarded the prize for his work on the development and control of the immune system.